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Home , Adenylate cyclase toxin, Bordetella

pertussis adenylate cyclase translocation across a tethered

Rémi Venezianoa, Claire Rossib, Alexandre Chenalc, Jean-Marie Devoissellea, Daniel Ladantc,1, and Joel Chopineaua,d,1

aEquipe Matériaux Avancés pour la Catalyse et la Santé, Unité Mixte de Recherche 5253, Centre National de la Recherche Scientifique-Ecole Nationale Supérieure de Chimie de Montpellier-Université Montpellier 2-Université Montpellier 1, Institut Charles Gerhardt Montpellier, 34296 Montpellier Cedex 5, France; bCentre National de la Recherche Scientifique Formation de Recherche en Evolution 3580, Université de Technologie de Compiègne, 60205 Compiègne Cedex, France; cCentre National de la Recherche Scientifique Unité Mixte de Recherche 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, 75724 Paris Cedex 15, France; and dUniversité de Nîmes, 30021 Nîmes Cedex, France

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved November 8, 2013 (received for review July 17, 2013)

Numerous bacterial can cross biological membranes to ATP-cyclizing, CaM-activated catalytic domain (AC) is located reach the cytosol of mammalian cells, where they exert their cy- in the 400 amino-proximal residues, whereas the carboxyl-terminal totoxic effects. Our model toxin, the adenylate cyclase (CyaA) from 1,306 residues are responsible for the hemolytic phenotype of , is able to invade eukaryotic cells by translo- B. pertussis (17–20). cating its catalytic domain directly across the plasma membrane The C-terminal “” moiety contains, between resi- of target cells. To characterize its original translocation process, dues 500 and 750, several hydrophobic segments that are pre- we designed an in vitro assay based on a biomimetic membrane dicted to adopt alpha-helical structures and to insert into model in which a tethered lipid bilayer (tBLM) is assembled on an membranes to create the cation-selective pores responsible for the hemolytic activity (20, 21). The C-terminal part of the mol- amine-gold surface derivatized with (CaM). The assem- – bled bilayer forms a continuous and -impermeable bound- ecule (RD; residues 1,000 1,706) is involved in toxin binding to a specific cellular receptor (CD11b/CD18) (22, 23). This domain ary completely separating the underlying calmodulin (trans side) consists of approximately 40 copies of a -binding, - from the medium above (cis side). The binding of CyaA to the tBLM and aspartate-rich nonapeptide repeat (residues 1,014–1,613) is monitored by surface plasmon resonance (SPR) spectroscopy.

characteristic of a large family of bacterial cytolysins known as BIOCHEMISTRY CyaA binding to the immobilized CaM, revealed by enzymatic ac- repeat-in-toxin (RTX) toxins (11, 13, 24, 25). tivity, serves as a highly sensitive reporter of toxin translocation The CyaA toxin is synthesized as an inactive precursor, pro- across the bilayer. Translocation of the CyaA catalytic domain was CyaA, which is converted into the active toxin form (CyaA) on found to be strictly dependent on the presence of calcium and also specific acylation of two residues (26, 27). Then CyaA is on the application of a negative potential, as shown earlier in eukary- secreted across the bacterial envelope by a dedicated type I se- otic cells. Thus, CyaA is able to deliver its catalytic domain across a cretion machinery and binds to the CD11b/CD18 integrin biological membrane without the need for any eukaryotic compo- expressed by a subset of leukocytes including , mac- nents besides CaM. This suggests that the calcium-dependent CyaA rophages, and dendritic cells (22, 28–30). However, CyaA can translocation may be driven in part by the electrical field across the also invade a wide variety of cells that do not express this re- membrane. This study’s in vitro demonstration of toxin transloca- ceptor, albeit with a lower efficiency (19, 31–35). tion across a tBLM provides an opportunity to explore the molecular The most unique property of CyaA is its capability to deliver its mechanisms of protein translocation across biological membranes N-terminal catalytic domain directly across the plasma membrane in precisely defined experimental conditions. of the eukaryotic target cells, a process that occurs independently of the CD11b/CD18 receptor (11–13). It is believed that CyaA first adenylate cyclase activity | synthetic biomembrane | toxin internalization Significance ransport of protein across the is a complex Tprocess that usually involves multipart translocation ma- Many bacterial toxins can cross biological membranes to reach chineries. Many protein toxins from poisonous plants or from the cytosol of mammalian cells, although how they pass pathogenic are able to penetrate into the cytosol of their through a lipid bilayer remains largely unknown. Bordetella target cells where they exert their toxic effects. Some of these pertussis adenylate cyclase (CyaA) toxin delivers its catalytic toxins exploit the endogenous cellular machinery of domain directly across the cell membrane. To characterize this and intracellular sorting to gain access to the cell cytosol, but unique translocation process, we designed an in vitro assay others carry their own translocation apparatus (1–4). These lat- based on a tethered lipid bilayer assembled over a biosensor ter toxins provide a unique opportunity to analyze the molecular surface derivatized with calmodulin, a natural activator of the mechanisms and the physicochemical principles underlying poly- toxin. CyaA activation by calmodulin provided a highly sensi- peptide transport across biological membranes. Studies combining tive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent structural, biochemical, and electrophysiological approaches have but independent of any additional eukaryotic protein. This bio- begun to unravel the various strategies developed by these toxins – mimetic membrane will permit in vitro studies of protein trans- to deliver their catalytic moieties across the cell membranes (5 10). location in precisely defined conditions. The (CyaA) produced by Bordetella pertussis , the causative agent of , is one of the Author contributions: D.L. and J.C. designed research; R.V., C.R., and A.C. performed re- few known toxins able to invade eukaryotic cells through a mech- search; J.-M.D. and D.L. contributed new reagents/analytic tools; R.V., D.L., and J.C. ana- anism of direct translocation across the plasma membrane of the lyzed data; and R.V., D.L., and J.C. wrote the paper. target cells (11–13). CyaA is an essential of B. The authors declare no conflict of interest. pertussis that is secreted by virulent bacteria and able to enter into This article is a PNAS Direct Submission. eukaryotic cells, where, on activation by endogenous calmodulin 1To whom correspondence may be addressed. E-mail: [email protected] or joel. (CaM), it catalyzes high-level synthesis of cAMP, which in turn [email protected]. – alters cellular physiology (14 16). CyaA is a 1,706-residue-long This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bifunctional protein organized in a modular fashion (Fig. 1A); the 1073/pnas.1312975110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1312975110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 immobilized CaM. With this highly sensitive assay, translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and application of a negative trans- membrane potential, in agreement with previous studies on eu- karyotic cells (36). Our results demonstrate that CyaA does not require any specific eukaryotic components apart from CaM to translocate across a membrane. They also suggest that the catalytic domain may be electrophoretically transported across the bilayer in a calcium-dependent manner. This study provides a direct in vitro demonstration of a toxin translocation across a tBLM (41) and suggests that the biomimetic tBLM/CaM structure may be a useful tool for characterizing the molecular mechanisms of protein translocation across biological membranes under pre- cisely defined conditions. Results Biomimetic Membrane Assembly. The tBLM/CaM structure was assembled in a homemade SPR cell comprising a gold- coated glass slide covered by a Teflon chamber (∼1 mL) with inlet and outlet tubes and mounted on an SPR optical bench instrument using the Kretschmann configuration (Fig. 1C and Fig. S1). The tBLM/CaM structure was obtained in two steps as described previously (40), using SPR spectroscopy to follow the grafting of the successive molecular layers (Fig. 2, Table S1, and Fig. S2). In the first step, the gold surface was amino-grafted by self-de- position of 2-aminoethanethiol on which CaM was covalently coupled through 1-ethyl-3-(3-dimethylaminopropyl) carbodii- mide (EDC) activation. The immobilized CaM was functional in binding CyaA, as demonstrated by SPR spectroscopy, and in stimulating its adenylate cyclase enzymatic activity (Fig. 2A; Table S1, experiment A; and Fig. S2A). In the second step, the tBLM was assembled over the CaM layer by incubating an L-α-phosphatidylcholine from egg yolk Fig. 1. Principle of CyaA translocation assay on tBLM/CaM assembly. (A) (egg-PC) vesicle suspension doped with 5% 1,2-distearoyl-sn- Scheme of CyaA toxin structure showing the three major domains: the cat- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- alytic domain, AC; the hydrophobic region, H, responsible for insertion of 3400]-succinimidyl propionate (DSPE-PEG3400-SPA) lipopolymer + CyaA into the membrane; and the Ca2 -binding, RTX-containing domain, for 1 h at 25 °C to covalently anchor the vesicles to the amine- RD. (B) Schematic illustration of the approach used to monitor CyaA trans- coated gold surface. The Teflon cell was then flushed extensively location across the tBLM. (C) Schematic representation of the SPR sample cell with HBS/EGTA buffer (20 mM Hepes-Na, 0.15 M NaCl, and cross-section and tBLM/CaM construction. 2 mM EGTA; pH 7.4), to remove unlinked vesicles and favor the formation of a continuous planar bilayer. The optical thickness of the tBLM assembled over the immobilized CaM was repro- inserts its hydrophobic segments into the plasma membrane and ducibly ∼54 ± 2 Å (Fig. 2B; Table S1, experiment B; and Fig. then delivers its catalytic domain across the plasma membrane S2B), a value consistent with the formation of a planar lipid bi- B into the cell cytosol (19, 31, 32) (Fig. 1 ). Previous studies have layer, as reported previously (40, 42). The fluidity and continuity shown that the translocation process is dependent on the tem- of the bilayer were confirmed by fluorescence recovery after perature (occurring only above 15 °C), the membrane potential of photobleaching experiments, as described in our previous report the target cells, and the presence of calcium ions in the mM range (40). The tBLM could be efficiently removed by washing with (32, 36). Inside the cell, on binding to CaM with a subnanomolar HBS/EGTA buffer containing a nonionic detergent (0.5% Triton affinity, CyaA is stimulated by more than 1,000-fold and exhibits X-100) as indicated by a large decrease in the SPR signal (Fig. k > −1 a high catalytic rate ( cat 2,000 s ) to produce supraphysiologic 2B; Table S1, experiment B; and Fig. S2B). levels of cAMP (12, 19, 37). After washing out the bilayer, we verified that the immobilized How the hydrophilic CyaA catalytic domain of approximately CaM was still functional in binding CyaA and activating its en- 400 residues is able to pass across the hydrophobic barrier of the zymatic activity (Fig. 2B; Table S1, experiment B; and Fig. S2B). plasma membrane remains largely unknown, and whether spe- This also indicated that nonionic detergent treatment did not cific eukaryotic and/or cell membrane components are affect CyaA binding and its activation by the tethered CaM. involved in this process is also unclear (19, 32, 35, 38, 39). To characterize the molecular mechanisms of CyaA translocation CyaA Binding to and Translocation Across the Biomimetic Membrane. across the membrane, we performed a functional in vitro assay When CyaA was diluted (200 nM) in HBS/CaCl2 buffer (20 mM that exploits a recently designed biomimetic membrane assembly Hepes-Na, 0.15 M NaCl, and 2 mM CaCl2; pH 7.4) and injected composed of a bilayer membrane (tBLM) tethered over an on the top of the tBLM, it efficiently bound to the membrane, as amino-grafted gold surface derivatized with CaM (40). This demonstrated by the increased SPR signal (Fig. 2C; Table S1, multilayer biomimetic assembly exhibits the fundamental feature experiment C; and Fig. S2C). After an extensive wash with HBS/ of an authentic biological membrane in creating a continuous, EGTA buffer, a large fraction of the bound CyaA remained yet fluid phospholipidic barrier between two distinct compart- attached to the tBLM (amounting to 110 ± 10 ng/cm2), likely ments: a cis side, corresponding to the extracellular milieu, and as a result of insertion of its hydrophobic segments in the a trans side, marked by the cytosolic protein CaM (Fig. 1C). We bilayer. After washing with HBS/EGTA buffer + 0.5% Triton monitored the binding of CyaA to the tBLM by surface plasmon X-100, the SPR signal again decreased significantly (Table S1, resonance (SPR) spectroscopy, and detected the translocation of experiment C and Fig. S2C) to an optical thickness of ∼8Å, the catalytic domain across the bilayer by CyaA activation by the close to the 6 Å signal found in the previous experiment with

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1312975110 Veneziano et al. Downloaded by guest on September 26, 2021 Fig. 2. SPR monitoring of tBLM/CaM construction and CyaA translocation. The optical thicknesses recorded at the different stages of the tBLM/CaM con- struction and subsequently, during CyaA binding and translocation events, were measured by SPR (SI Materials and Methods). The numbers above the bars indicate the AC activity (in nmol/min) measured on the chip after the final wash with Triton X-100 (0.5%). The data are extracted from the traces shown in Fig. S2. The experiments are described in detail in the legend of Fig. S2.(A) Optical thicknesses measured after, sequentially, (i) CaM coupling to the cysteamine-

coated gold substrate (+ CaM), (ii) CyaA binding in HBS/CaCl2 buffer (+CyaA), and (iii)afinal wash with 0.5% Triton X-100 (+ TX-100). (B) Optical thicknesses measured after (i) CaM coupling (+ CaM) as above, (ii) bilayer membrane tethering (+ tBLM), (iii) tBLM removal by Triton X-100 wash (+ TX-100), (iv) CyaA

binding in HBS/CaCl2 (+ CyaA), and (v)afinal wash with 0.5% Triton X-100 (+ TX-100). (C) Optical thicknesses measured after (i) CaM coupling (+ CaM), (ii) bilayer membrane tethering (+ tBLM), (iii) CyaA binding in HBS/CaCl2 (+ CyaA), and (iv)afinal wash with 0.5% Triton X-100 (+ TX-100). (D) Optical thicknesses measured after (i) CaM coupling (+ CaM), (ii) bilayer membrane tethering (+ tBLM), (iii) CyaA binding in HBS/CaCl2 (+ CyaA), and (iv) application of voltage (−80 mV) for 5 min, followed by a final wash with 0.5% Triton X-100 (+ TX-100).

tBLM only (Table S1, experiment B). This finding suggests that the tBLM was washed with Triton X-100, the enzymatic activity most of the membrane-bound CyaA had been washed away as bound to the immobilized CaM was measured. Enzymatic ac- well, and indeed, only background levels of enzymatic activity tivity bound to the tethered CaM was strictly dependent on the BIOCHEMISTRY were detected on the surface of the SPR chip. Taken together, application of a negative potential across the membrane (Fig. 3). fi these results indicate that although CyaA had ef ciently bound These data are in excellent agreement with the voltage depen- the tBLM, its catalytic domain could not interact with the immo- dence of the CyaA intoxication of myocytes reported by Otero bilized CaM and thus was not retained on the chip on membrane et al. (36). removal. Thus, CyaA did not translocate under these experi- Of note, if after CyaA binding to tBLM (obtained in the pres- mental conditions. Based on the finding of Otero et al. (36) that a membrane ence of calcium), HBS/EGTA buffer was added before application potential is critical for CyaA intoxication of myocytes, we ex- of the electric potential, then no enzymatic activity was detected plored whether CyaA translocation could be achieved in our on the chip. This indicates that translocation of the CyaA catalytic biomimetic device after application of an electrical potential domain is strictly dependent on the presence of calcium in the mM across the tethered bilayer. In this set of experiments, CyaA was range (Fig. 3, Inset), as previously observed on various eukaryotic again allowed to bind to and insert into the tBLM as described cells (18–20, 30, 32, 35, 36, 38). Taken together, these data above (Fig. 2D; Table S1, experiment D; and Fig. S2D). After the unbound CyaA was washed with HBS/CaCl2 buffer, an electrical potential of −80 mV was applied for 5 min between the gold surface and an Ag wire (serving as a reference electrode con- nected to ground) connected to the bulk medium above the tBLM (i.e., between the trans and cis sides of the membrane); following convention, the bulk medium potential was set to 0 mV. Current recordings of the tBLM with or without bound CyaA at different voltages (Fig. S3) indicate that the tBLM created a good ion-impermeable barrier between the trans and cis compartments. After the membrane was washed out with Triton X-100 (i.e., HBS/EGTA + 0.5% Triton X-100) the SPR signal decreased (Fig. 2D; Table S1, experiment D; and Fig. S2D) to a value (∼200 ng/cm2) significantly higher than that found without the application of electrical potential (80 ng/cm2; Table S1). This finding suggests that a fraction of the CyaA protein was retained on the SPR chip. This was confirmed by enzymatic assays, which Fig. 3. Voltage and calcium dependence of CyaA translocation across tBLM. CyaA (200 nM) was injected on top of the tBLM/CaM assembly in HBS/CaCl2 detected a high level of adenylate cyclase activity on the chip 2+ (Fig. S4). We conclude that on application of a negative potential buffer. After extensive washing with HBS/CaCl2 (black triangle; 2 mM Ca across the bilayer, the catalytic domain of CyaA was translocated on the cis side) or HBS/EGTA (black square; 2 mM EGTA on the cis side), the across the tBLM and could interact with the immobilized CaM to indicated potentials were applied for 5 min. The potential in bathing me- dium (i.e., on the cis side) was set at 0. After extensive washing in HBS/EGTA, acquire its enzymatically active form. the tBLM was removed by washing with 0.5% Triton X-100 in HBS/EGTA, and AC enzymatic activity was measured. (Inset) CyaA binding to tBLM/CaM was Voltage and Calcium Dependence of CyaA Translocation Across the fi carried out in HBS/CaCl2 buffer as above. The membrane was then washed tBLM. We carried out additional experiments to measure the ef - with HBS buffer containing the indicated calcium concentrations, and a −80- cacy of the translocation process as a function of membrane po- mV potential was applied for 5 min. The AC activity was measured after tential. CyaA was bound to the tBLM as described above, and after membrane removal as above, and expressed as a percentage of AC activity

the unbound CyaA was washed out, various electrical potentials, measured with 2 mM CaCl2. Values are mean ± SEM from at least three ranging from −80 mV to +50 mV, were applied for 5 min. After independent measurements for each condition.

Veneziano et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 demonstrate that our in vitro translocation assay on tBLM is −80 mV to +50 mV, and was still forming a continuous lipidic able to reproduce the fundamental properties of CyaA intox- barrier impermeable to small proteins like trypsin. However, at ication of target cells, that is, its calcium and membrane potential a higher positive potential (+80 mV), the bilayer integrity ap- dependency. parently was partially impaired, allowing nonspecific access of proteins (CyaA or trypsin) to the underlying trans compartment. Probing tBLM Integrity with a Protection Assay. To further validate the in vitro translocation assay, we performed control mAb 3D1 Blocks CyaA Translocation Across the tBLM. Gray et al. (33) experiments to verify that application of a voltage across the previously reported that a monoclonal , mAb 3D1, membrane had not impaired the integrity of the tBLM by cre- which recognizes an epitope located between residues 373 and ating “holes” through which CyaA could have accessed the 399 of CyaA at the C-terminal end of the catalytic domain, does immobilized CaM. For this, we conducted a protease protection not affect the adenylate cyclase activity of the toxin or its capacity assay similar to that used to measure CyaA translocation into to bind to target cells, but does inhibit delivery of the catalytic erythrocytes or other cells (18–20, 32). First, CyaA was bound to domain into the cytosol of target cells (erythrocytes or J774A.1 the tBLM in HBS/CaCl2 buffer, and membrane polarization was cells). Thus, we tested the effect of mAb 3D1 in our in vitro applied for 5 min to trigger translocation of the catalytic domain translocation assay. We found that the addition of a molar excess across the tBLM. Then a solution of trypsin (20 nM in HBS/ of mAb 3D1 to the CyaA solution before injection into the construction did not prevent binding to tBLM, but did signifi- CaCl2 buffer) was injected, followed by incubation for 5 min at 25 °C, then extensive washing with a solution containing an ex- cantly diminish translocation of the catalytic domain, as dem- cess of soybean trypsin inhibitor (40 nM in HBS/CaCl ) to stop onstrated by the AC activity bound to the immobilized CaM 2 fi fi proteolysis. After the tBLM was washed out with Triton X-100 (Fig. 5). This nding con rms that our in vitro translocation assay (in HBS/EGTA buffer), AC enzymatic activity was measured closely mimics the biological process occurring on living cells. as before. AC activities measured with or without trypsin treatment were Nonacylated ProCyaA Cannot Deliver Its Catalytic Domain Across the ’ very similar at all tested potentials from −80 mV to +50 mV (Fig. Tethered Bilayer. It is well known that CyaA s invasive capacity 4). However, when a higher positive potential (+80 mV) was depends on its acylation on the lysine residues K860 and K983 applied, a significant fraction of AC activity was detected in the (26, 27). Thus, we tested nonacylated proCyaA in our in vitro absence of trypsin treatment, but not after trypsin treatment tBLM/CaM translocation assay. ProCyaA and CyaA bound to the tBLM to a similar extent, as detected by SPR signals (Fig. 6); (Fig. 4). We conclude that this high positive potential did not − trigger CyaA translocation, but rather partially impaired the in- however, after application of a 80-mV potential in the presence tegrity of the bilayer, allowing access of CyaA to the immobilized of CaCl2, only background levels of enzymatic activity were CaM and, subsequently, access of trypsin as well. detected on the chip with proCyaA, in contrast to the levels seen We also verified that when the trypsin treatment was applied with CyaA. This result demonstrates that the acylation of CyaA after CyaA binding but before the application of voltage (i.e., is critical for conferring the ability to deliver its catalytic domain before translocation), no AC activity was detected on the chip. across the tBLM, similar to that observed on eukaryotic cells. fi This nding indicates that after binding and insertion of CyaA Discussion into the tBLM, the catalytic domain is readily accessible to trypsin degradation on the “external” side of the bilayer. CyaA is unique among the bacterial toxins for its capacity to Finally, we verified that the AC enzyme bound to the immo- deliver its catalytic domain (AC) into the cytosol of target cells bilized CaM after translocation was fully degraded by trypsin directly across the plasma membrane. To characterize this orig- when the tBLM was previously removed by washing with Triton inal translocation process, we describe here an in vitro assay based on a biomimetic membrane model consisting of a tBLM X-100. Based on these results, we conclude that the bilayer was assembled over a SPR biosensor surface derivatized with CaM. not altered by application of electrical potentials ranging from The tBLM forms a continuous, protein-impermeable boundary that fully insulates the immobilized CaM (trans side) from the medium above (cis side). The binding of CyaA to the tBLM can be monitored by SPR spectroscopy, whereas binding of CyaA to the shielded, immobilized CaM serves as a highly sensitive re- porter of toxin translocation across the bilayer. We demonstrated with this in vitro tBLM/CaM system, that the translocation of the AC domain was strictly dependent on the presence of calcium and on application of a negative electrical potential across the membrane, in excellent agreement with previous studies of Otero et al. (36) performed on living eukaryotic target cells. The overall efficiency of translocation (approximately 80% of total tBLM-bound CyaA) observed in vitro compares favorably with that measured previously on sheep Fig. 4. Probing the tethered bilayer integrity by a protease protection as- erythrocytes (32, 43). Furthermore, no translocation was ob- say. CyaA (200 nM) was injected onto tBLM/CaM in HBS/CaCl2 buffer. After served with the nonacylated proCyaA protein or after pre- extensive washing with HBS/CaCl2 buffer, the indicated potentials were incubation of CyaA with mAb 3D1, which had been previously applied for 5 min as described in Fig. 3 with 2 mM Ca2+ on the cis side. For + shown to block the entry of CyaA into eukaryotic cells (30, 33, the voltage trypsin condition, 20 nM trypsin (in HBS/CaCl2) was injected in 38). This finding further supports our contention that our in vitro the device and incubated for 5 min, followed by the addition of 40 nM assay captures the essential features of the authentic physiolog- soybean trypsin inhibitor (STI). For the trypsin + voltage condition, CyaA (200 nM) was bound to tBLM as above, but the trypsin incubation (20 nM in HBS/ ical processes occurring with living cells. CaCl for 5 min, followed by the addition of STI) was carried out before Importantly, our data show that no additional eukaryotic com- 2 ponents are needed for the transfer of the catalytic domain across application of the electrical potential. After extensive washing in HBS/CaCl2, the tBLM was removed in all samples with 0.5% Triton X-100 (in HBS/EGTA the tBLM, indicating that the CyaA protein contains all of the buffer), and AC enzymatic activity was measured. As a control (voltage + elements required for this process. Thus, translocation of CyaA Triton X-100 + trypsin), CyaA was bound to tBLM, a −80-mV potential was catalytic domain across the tBLM depends only on calcium and applied as above, and trypsin proteolysis was carried out after removal of membrane potential, which could provide the driving force for tBLM by washing with Triton X-100. Values are mean ± SEM from at least polypeptide transport across the membrane via an electrophoretic three independent measurements for each condition. process (36, 43). However, we cannot exclude the possibility that

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1312975110 Veneziano et al. Downloaded by guest on September 26, 2021 The precise molecular mechanisms by which bacterial toxins can cross biological membranes to reach the cytosolic compart- ment of mammalian cells remain largely unknown (1, 2). Re- constitution of toxin translocation across synthetic model membranes is crucial to better characterize the structural and biophysical principles at work in this process, but designing ef- ficient in vitro systems for that purpose is challenging. The most effective approaches to date involve electrophysiological studies Fig. 5. mAb 3D1 blocks CyaA translocation across the tBLM. CyaA (200 nM) on planar lipid bilayers. Indeed, many toxins are known to form ion-conductive pores in model membranes (5, 6), and it has been was injected above the tBLM/CaM in HBS/CaCl2 as in Fig. 2 (calcium or EGTA, + shown that these pores can be transiently blocked by their cat- or 3D1 CyaA) after preincubation (20 min at 25 °C) with mAb 3D1 (200 cis nM). After extensive washing with HBS/CaCl2 (calcium or 3D1 + CyaA) or alytic subunits as they pass through the pore from the side to HBS/EGTA (EGTA), a potential of −80 mV was applied for 5 min. Then tBLM the trans side of the membrane (7–9). This highly sensitive was removed with 0.5% Triton X-100 in HBS/EGTA buffer, and AC enzymatic method has been instrumental to the characterization of many activity was measured. For each condition, the quantity (ng/cm2) of protein structural and functional aspects of toxin translocation; however, bound to the tBLM was determined by SPR spectroscopy. Values are mean ± direct demonstration of the passage of the catalytic subunit SEM from at least three independent measurements for each condition. from the cis compartment to the trans compartment of the membrane is difficult, given the very low quantities of molecules delivered (7). the tight association of CyaA with the immobilized CaM may More recently, Fisher et al. (10) developed a droplet-interface contribute in part to translocation of the AC across the bilayer. bilayer (DIB) approach to monitoring the transfer of anthrax Indeed, it has been shown that for several toxins (e.g., diphtheria toxin across a model membrane separating two submicroliter toxin, anthrax lethal factor, anthrax edema factor), the entry aqueous droplets. In this approach, a lipid bilayer membrane is process is facilitated in vivo by specific interactions with dedicated formed at the interface between two droplets connected to cytosolic factors, such as the coatomer I complex, Hsp90, or thi- electrodes. Because the droplets can be moved into contact or oredoxin reductase, which recognize specific sequences separated by a micromanipulator, it is possible to follow, with an within the toxin polypeptide chains (44–47). These interactions exquisite sensitivity, the physical transit of material (e.g., pro- likely contribute to pull the catalytic chains of these toxins through teins) from one droplet to the other through a protein pore. Such

the membrane through a Brownian ratchet-like mechanism (48), a system might be of general interest for many other toxins. BIOCHEMISTRY and could aid toxin refolding within the cytosol. CaM likely can We present here a novel approach to study protein trans- play such a chaperone-like role for CyaA (49). However, it has location in tBLM. These biomimetic models have been widely been shown that mutant CyaA toxins exhibiting low affinity for used to characterize protein–membrane associations, binding of CaM are still able to efficiently deliver their AC domain into the ligands to cellular receptors incorporated in the supported pla- of cells (50), suggesting that a high affinity of CyaA for nar bilayers, and molecular interactions in cell adhesion pro- CaM might not be critical for CyaA translocation. cesses (41, 42, 52, 53); however, up to now, few studies have been CyaA is known to invade a wide variety of cell types in vivo, devoted to protein transport across reconstituted bilayers, owing fi albeit with varying ef cacies and possibly through different to various technical difficulties. Our tBLM/CaM technique is pathways, and to target primarily innate cells particularly well adapted for characterizing protein translocation that express the CD11b/CD18 integrin receptor to which its across membranes, allowing for direct detection of the passage of fi binds with high af nity (22, 29, 30, 38, 39). CyaA binds with polypeptides from the cis side to the trans side of the membrane. fi much lower af nity to cells that do not express this receptor (19, This is clearly an advantage over the planar lipid bilayer and DIB – 26, 32 34, 36, 43, 51), but whether the translocation process per approaches discussed above. However, if the tBLM is fully im- se is modulated by the CD11b/CD18 receptor remains unknown permeable to protein, its electrical properties cannot be com- (30, 38, 39). A more sophisticated biomimetic tBLM/CaM con- pared with those of planar lipid bilayers or DIB techniques, struction harboring the CD11b/CD18 receptor inserted in the which remain unrivaled for studying channels or pore-forming membrane might be feasible in the future. proteins. The in vitro approach elaborated herein may be ap- plicable to other toxins that associate with CaM, such as the anthrax edema factor, and also could be adapted for virtually any toxins that target specific cytosolic factors (1). These biomimetic constructions should provide opportunities to explore the mo- lecular mechanisms of protein translocation across biological membranes in precisely defined in vitro conditions. Materials and Methods Proteins were produced and purified in and stored at −80 °C. For tBLM construction, CaM (140 nM in HBS/EGTA buffer: 20 mM Hepes-Na, 0.15 M NaCl, and 2 mM EGTA; pH 7.4) was covalently linked to the amine- grafted surface in the presence of EDC (350 nM). The CaM surface coverage thus obtained was approximately 35 ng/cm2 (40). tBLM egg-PC/DSPE- Fig. 6. ProCyaA cannot deliver its catalytic domain across tBLM. CyaA or PEG3400-SPA (95/5 wt/wt ratio) was achieved by injection of a small uni- proCyaA (200 nM) was injected either onto the immobilized CaM without lamellar vesicle suspension (1 mg/mL in HBS/EGTA buffer) on top of the CaM tBLM ( CaM) or onto the tBLM/CaM construction as in Fig. 2. After extensive layer, followed by incubation at room temperature for 1 h.

washing with HBS/CaCl2,a–80-mV potential was applied for 5 min on the CyaA and proCyaA were incubated in the biomimetic system at 200 nM – tBLM/CaM assembly or not applied, as indicated. For each condition, the (with 2 mM CaCl2). For the mAb CyaA complex, preincubation with CyaA quantity of protein bound to the tBLM or CaM was determined by SPR and antibody (1:1 ratio) was performed for 20 min at 25 °C before injection spectroscopy, after which the SPR surfaces were washed extensively with into the SPR sample cell. SPR measurements were performed with a home- 0.5% Triton X-100 in HBS/EGTA buffer and AC enzymatic activity was mea- made optical setup in a Kretschmann configuration (Fig. S1). The gold- sured. Protein amount (black bars) and AC activity (white bars) are expressed coated glass slide was vertically assembled with a Teflon sample cell (total in percentages, taking the protein binding and AC activity measured on volume, 925 μL; gold surface, 1.54 cm2). The optical thicknesses and amounts immobilized CaM (without tBLM) as 100%. Values are mean ± SEM from at of lipids or proteins bound on the surfaces were determined as described in least three independent measurements for each condition. SI Materials and Methods.

Veneziano et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 The membrane translocation assay was performed in the SPR cell using two The enzymatic activity assay was performed as described previously (40). In electrodes for application of a membrane potential. The gold functionalized brief, tBLM was removed with Triton X-100, after which 0.5 mL of AC buffer glass slide was set as the trans electrode, and the cis Ag electrode was bathed containing ATP (2 mM) and pyrophosphatase (2 U/mL) was added and in- in buffer (Fig. 1C and Fig. S1). Potentials were applied using a Bio-Logic BLM- cubated for 10 min. Pi release was measured with a Pi ColorLock Kit 120 amplifier equipped with a 1-GΩ head, and the signal was attenuated (Innova Bioscience). by an 8-pole low-pass filter (AF-180; Bio-Logic). The potential was applied for 5 min, and the current was monitoredwithanoscilloscope(TDS3012; ACKNOWLEDGMENTS. We thank Agnes Ullmann for a critical reading of the Tektronix). manuscript and Jean-Yves Le Guennec for fruitful discussions. The project was supported by the Institut Pasteur and by the Centre National de la The trypsin proteolysis assay was initiated with trypsin (20 nM) added into Recherche Scientifique (Unité Mixte de Recherche 5253, Institut Charles Ger- the SPR sample cell for 5 min and stopped by the addition of trypsin inhibitor hardt Montpellier and Unité Mixte de Recherche 3528, Biologie Structurale (40 nM). des Processus Cellulaires et Maladies Infectieuses).

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